Evaluation of laboratory assays for screening antibody to hepatitis C virus

An evaluation study, involving 11 screening methods for the detection of antibody to hepatitis C virus (anti-HCV) and a panel of 500 serum samples, was performed. Samples were tested by all 11 methods, and those showing reactivity in at least one method were studied by a combination of supplemental assays (recombinant immunoblot assays, first and second generation; neutralization test for anti-c100; synthetic peptide immunoblot assay; recombinant multi-dot immunoassay) and classified as positive (110 samples), indeterminate (4 samples), or negative (386 samples) on the basis of the results obtained. Second- generation recombinant methods performed better on positive samples than first-generation assays or synthetic peptide-based methods (99.1- 100% correlation vs. 64.5-85.5% and 93.6-99.1%, respectively), whereas the latter showed higher correlations on negative samples than recombinant assays (97.4-99.7% vs. 82.4-93%). Further investigations, using broad panels of indeterminate samples from blood donors, should be done, however, before synthetic peptide-based methods are recommended for blood bank screening. Reactivity of samples must be confirmed by one supplemental test in all cases before the donor is informed. In some cases, it may require the use of two or more different tests to obtain definite conclusions.     

Normal and disrupted lumbar longitudinal ligaments: correlative MR and anatomic study

The posterior and anterior longitudinal ligaments of the lumbar spine appear on magnetic resonance (MR) images as thin lines of very low signal intensity in all spin-echo sequences. They cover the periphery of the outer fibers of the anulus fibrosus on sagittal images. The lumbar spine of 17 patients with 19 disk herniations was prospectively evaluated with MR imaging, and these findings were correlated with surgical findings. At surgery the posterior ligament was found to be disrupted in eight cases and intact in 11. Absence of a low-signal peripheral line around the herniated nucleus pulposus (HNP) was the most reliable sign of ligament rupture (no false-negative or false-positive findings). The peripheral line appeared to be interrupted in four cases, two of which were falsely positive. The two false-positive cases were related to a chemical shift artifact between epidural fat and the HNP. Presence of a normal and continuous peripheral line outlining the HNP excluded ligament disruption. The overall sensitivity for detecting disruption was 100%, and the specificity was 78%.     

Insulin‐like growth factor‐II is produced by,signals to and is an important survival factor for the mature podocyte in man and mouse

Podocytes are crucial for preventing the passage of albumin into the urine and, when lost, are associated with the development of albuminuria, renal failure and cardiovascular disease. Podocytes have limited capacity to regenerate, therefore pro‐survival mechanisms are critically important. Insulin‐like growth factor‐II (IGF‐II) is a potent survival and growth factor; however, its major function is thought to be in prenatal development, when circulating levels are high. IGF‐II has only previously been reported to continue to be expressed in discrete regions of the brain into adulthood in rodents, with systemic levels being undetectable. Using conditionally immortalized human and ex vivo adult mouse cells of the glomerulus, we demonstrated the podocyte to be the major glomerular source and target of IGF‐II; it signals to this cell via the IGF‐I receptor via the PI3 kinase and MAPK pathways. Functionally, a reduction in IGF signalling causes podocyte cell death in vitro and glomerular disease in vivo in an aged IGF‐II transgenic mouse that produces approximately 60% of IGF‐II due to a lack of the P2 promoter of this gene. Collectively, this work reveals the fundamental importance of IGF‐II in the mature podocyte for glomerular health across mammalian species. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

An international survey of EEG use in the neonatal intensive care unit

Objective: To examine the extent of EEG monitoring in neonatal intensive care units (NICUs), and to survey the level of experience and training of those using it. Study design: A web‐based survey, the link to which was circulated via e‐mail, personal contact, specialist societies and professional groups. Survey data were exported to SPSS for analysis. Results: In total 210 surveys were analysed; 124 from Europe, 54 from the US. Ninety percent of respondents had access to either EEG or aEEG monitoring; 51% had both. EEG was mainly interpreted by neurophysiologists (72%) whereas aEEG was usually interpreted by neonatologists (80%). Only 9% of respondents reported that they felt ‘very confident’ in their ability to interpret aEEG/EEG with 31% reporting that they were ‘not confident’. Half had received no formal training in EEG. Conclusion: Both aEEG and conventional EEG were used extensively in the NICUs surveyed for this study. Most of the survey respondents were not confident in their ability to interpret EEGs despite the fact that they used monitoring routinely. There is an urgent need for a structured and appropriately targeted training programme in EEG methodologies and EEG interpretation for neonatal intensive care unit staff.     

New method for analysing sensitivity distributions of electroencephalography measurements

In this paper, we introduce a new modelling related parameter called region of interest sensitivity ratio (ROISR), which describes how well the sensitivity of an electroencephalography (EEG) measurement is concentrated within the region of interest (ROI), i.e. how specific the measurement is to the sources in ROI. We demonstrate the use of the concept by analysing the sensitivity distributions of bipolar EEG measurement. We studied the effects of interelectrode distance of a bipolar EEG lead on the ROISR with cortical and non-cortical ROIs. The sensitivity distributions of EEG leads were calculated analytically by applying a three-layer spherical head model. We suggest that the developed parameter has correlation to the signal-to-noise ratio (SNR) of a measurement, and thus we studied the correlation between ROISR and SNR with 254-channel visual evoked potential (VEP) measurements of two testees. Theoretical simulations indicate that source orientation and location have major impact on the specificity and therefore they should be taken into account when the optimal bipolar electrode configuration is selected. The results also imply that the new ROISR method bears a strong correlation to the SNR of measurement and can thus be applied in the future studies to efficiently evaluate and optimize EEG measurement setups.     

Protease‐activated receptor 2 suppresses lymphangiogenesis and subsequent lymph node metastasis in a murine pancreatic cancer model

Protease‐activated receptor‐2 (PAR‐2) is a G protein‐coupled receptor that functions as a cell‐surface sensor for coagulation factors and other proteases associated with the tumour microenvironment. Pancreatic cancer cells express high levels of PAR‐2 and activation of PAR‐2 may induce their proliferation and migration. Interestingly, however, PAR‐2 expression is increased in stroma‐rich pancreatic cancer regions, suggesting a potential role of PAR‐2 in the tumour microenvironment. Here, we assessed the importance of PAR‐2 in the stromal compartment by utilizing an orthotopic pancreatic cancer model, in which tumour cells are PAR‐2‐positive, whereas stromal cells are PAR‐2‐negative. We assessed tumour weight and volume and analysed proliferation and (lymph)angiogenesis both in vivo and in vitro. We show that genetic ablation of PAR‐2 from the stromal compartment inhibits primary tumour growth, which is accompanied by reduced vascularization in primary tumours and reduced in tube formation of vascular endothelial cells in vitro. In contrast to smaller primary tumours, the number of lymph node metastases was increased in PAR‐2‐deficient animals, which was accompanied by an increased number of lymphatic vessels. In vitro tube‐formation assays show that PAR‐2 does not inhibit the intrinsic tube‐forming capacity of lymphatic endothelial cells, but that PAR‐2 actually inhibits cancer cell‐induced tube formation. Overall, stromal PAR‐2 thus plays a dual role in pancreatic cancer development by potentiating primary tumour growth but limiting lymphangiogenesis and subsequent lymph node metastasis. Our data identify a novel role of PAR‐2 in the tumour microenvironment and pinpoint PAR‐2 as a negative regulator of lymphangiogenesis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle

Skeletal muscle is an attractive target for somatic gene transfer of both acquired and inherited disorders. Direct injection of adenoviral vectors in the skeletal muscle leads to recombinant gene expression in a large number of muscle fibers. Transgene expression has been transient in most organs and associated with substantial inflammation when experiments are performed in adult immune competent mice. In this report, we utilize a variety of in vivo and in vitro models of T and B cell function to characterize the nature of the immune response to adenoviral vectors injected into murine skeletal muscle. Cellular immunity dependent on CD4+ and CD8+ T cells contributes to the loss of recombinant gene expression and the development of localized inflammation. Antigen specific activation of T cells occurs to both viral proteins and the reporter gene beta-galactosidase. Systemic levels of neutralizing antibody to the capsid proteins of the vector are also generated. Destructive immune responses responsible for loss of transgene expression are largely directed against beta-galactosidase in that transgene expression was stable when beta-galactosidase was eliminated as a neoantigen in mice transgenic for lacZ. A strategy to prevent the cellular and humoral immunity to this therapy was developed based on transiently ablating CD4+ T cell activation at the time of vector delivery. Encouraging results were obtained when vector was administered with one of several immune modulating agents including cyclophosphamide, mAb to CD4+ cells, and mAb to CD40 ligand. These studies indicate that cellular and humoral immune responses are elicited in the context of gene therapy directed to skeletal muscle with adenoviral vectors. Transient ablation of CD4+ T cell activation prevents the effects responses of the CD8+ T and B cells.